Non-invasive diagnostic imaging technology for mitochondria using radiolabeled lipophilic salts

ABSTRACT

The invention provides lipophilic salts, particularly, lipophilic salts comprising a pharmaceutically acceptable anion and at least one phosphonium or ammonium cation. In certain embodiments, the cation is labeled with one or more radioisotopes. The lipophilic salts of the invention exhibit an affinity for dysfunctional mitochondria, and are useful for the imaging of cardiovascular diseases and disorders. The invention also provides pharmaceutical compositions and methods of using the lipophilic salts.

This application claims the benefit of U.S. Provisional Application Ser.No. 60/354,563 filed Feb. 6, 2002, the teachings of which areincorporated herein by reference.

This invention was supported by National Institute of Health (NIH) GrantNo. CA92871. The United States government has certain rights to theinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention provides novel radiolabeled lipophilic salts,particularly radiolabeled lipophilic phosphonium and ammonium salts,which are capable of measuring mitochondrial surface potential (ΔΨm).This invention also provides pharmaceutical compositions comprising suchradiolabeled lipophilic salts. Additionally this invention providesimaging methods for identifying tissues or cells having aberrant levelsof mitochondrial activity by selectively localizing radiolabeledlipophilic salts of the invention into dysfunctional mitochondria. Theinvention also provides on-invasive methods for an early and sensitivedetection of tumor response to chemotherapy agents. The inventionfurther provides treatment methods comprising administration of a highenergy radiolabeled lipophilic salts to a patient, particularly patientssuffering from diseases or disorders associated with mitochondrialdysfunction.

2. Background

Measurement of the mitochondrial membrane potential (ΔΨm) provides thesingle most comprehensive reflection of mitochondrial bio-energeticfunction primarily because it directly depends on the proper integrationof diverse metabolic pathways that converge at the mitochondria.Numerous diseases are associated with mitochondria dysfunction,including cancer, cardiovascular and liver diseases, degenerative andautoimmune disorders as well as aging and new pathologies related tomitochondria are identified each year.

Alterations in ΔΨm is an important characteristic of a vast array ofpathologies that either involve suppressed (e.g., cancer) or enhancedapoptosis (e.g., HIV, degenerative disease) as well as >100 diseasesdirectly caused by mitochondrial dysfunction such as DNA mutations andoxidative stress (e.g., various types of myopaties).

There are SPECT imaging probes labeled with a technetium center whichare capable of accumulation in the mitochondria and the technetiumlabeled probes have been used for mitochondria based imaging techniques.There are a number of commercially available imaging probes that detecta given pathology using imaging agents such as [^(99m)Tc]MIBI, FDG.

[¹⁸F]FDG detects malignant lesion due to enhanced glucose metabolism.Further, as mentioned above, [¹⁸F]FDG is not able to differentiateneoplasm from inflammation. [¹⁸F]FDG is most effective imaging probe fortumor detection but poorly distinguishes neoplasm from inflammation,posing a frequent diagnostic challenge. In certain organs inflammation(e.g., tuberculosis) is a frequent pathologies among patients withsuspected malignant lesion. For example, >10% of pulmonary hot spotsindicated by [¹⁸F]FDG PET are inflammatory process rather than neoplasm,as proven by surgery. In other words, about 10% of lung patients with[¹⁸F]FDG PET indications may undergo unnecessary chest surgery, for adisease (inflammation) that otherwise can be treated in non-surgical andless costly and morbid approaches.

Current approaches for evaluation of efficacy of chemotherapy relies onalterations in tumor growth rate, a costly approach of limitedsensitivity which involves months of follow up, repeated visits inclinic, multiple radiographic scans and frequently a number of treatmentcycles.

Technetium labeled mitochondria imaging agents are hampered by severallimitations. More particularly, labeling a molecule with ^(99m)Tcrequires a conjugating moiety to complex the technetium ion such thatTc-based imaging agents have a high molecular weight which reduces thepermeability of the imaging agent in target areas. Further, technetiumimaging agents are imaged with SPECT which has relatively low spatialresolution and sensitivity when compared to comparable PET images.

There are technetium complexes, derivatives of [^(99m)Tc]annexin V, forapoptosis imaging by using SPECT. The novelty of the proposed[¹⁸F]phosphonium cations (PhCs) is that they detect the apoptoticprocess via a change in ΔΨm, whereas annexin V derivatives do so due tooverexpression of specific membrane proteins.

[^(99m)Tc]annexin V detects apoptosis due to externalization ofphosphatidylserine on the outer cytoplasm membrane. This event occurs atthe end of the apoptosis process when the fragmented cell is transformedinto clusters of molecules (apoptotic bodies). Shortly after theexternalization of phosphatidylserine (termed “eat me” phospholipids)the apoptotic bodies are phagocytized by neighboring cells. Therefore,detection of overexpression of phosphatidylserine is limited to a narrowtime window which may last a few days only. Furthermore, the time ofappearance and the duration of this window may vary among differentchemotherapy agents and subjects.

The collapse of ΔΨm is the point of no return of the apoptotic process.Therefore, the collapse of ΔΨm affords the earliest time point to detectapoptosis, rather the last event as in the case of annexin V, and thecollapse persists independent of time.

Current approached for the evaluation of myocardial perfusion andviability have several limitations, including masking of myocardialactivity by high accumulation in the organs adjacent to the heart(Th-201, [^(99m)Tc]MIBI) and short half-life of the isotope([¹³N]-ammonia and ⁸²Rubidum), thus limited to PET centers with anon-site cyclotron.

It would be desirable to have a family of lipophilic salts which have anaffinity for mitochondria, particularly mitochondria undergoing aberrantactivity.

SUMMARY OF THE INVENTION

In view of the high incidence of cancer cases (˜1.3 million per year inthe USA), the high frequency of chemotherapy applications and the lowfrequency of successful chemotherapy, there is an urgent need for anon-invasive imaging probe of rapid and sensitive assessment of tumorresponse to treatment. The need for diagnostic means in oncology is bestexemplified by the rapid transition of [¹⁸F]FDG PET from aninvestigational to a preferred diagnostic tool for tumor detectionwithin a few years.

There also exists a great need to diagnose and image cardiovasculardiseases and disorders, many of which are associates with mitochondrialdysfunction. Thus there is also an urgent need for non-invasive imagingprobes for rapid and sensitive measurement of cardiac uptake of imagingagents having an affinity for dysfunctional mitochondria for the imagingof cardiovascular diseases such as myocardial perfusion.

The invention provides novel lipophilic salts, particularly lipophilicsalts comprising a pharmaceutically acceptable anion and at least onephosphonium or ammonium cations according to Formula I, andpharmaceutical compositions comprising cations of Formula I and at leastone pharmaceutically acceptable carrier or excipient. Preferredlipophilic salts of the invention exhibit high affinity mitochondria,particularly dysfunctional mitochondria with enhanced or suppressedactivity.

The present invention provides salts comprising at least onepharmaceutically acceptable anion and at least one cation according toFormula I

wherein

E is phosphorus or nitrogen; and

X¹, X², X³, and X⁴ are independently selected from the group consistingof Ar and R, wherein at least one of X¹, X², X³, and X⁴ is an Ar group;

Ar is optionally substituted aryl, optionally substituted heteroaryl,and optionally substituted aralkyl; and

R is optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted haloalkyl,optionally substituted cycloalkyl, optionally substituted aralkyl,wherein at least one occurrence of R comprises at least oneradioisotope.

The salts of the invention which comprise a cation of Formula I aresuitable for use in imaging or assessment, particularly PET or SPECTimaging, of mitochondrial dysfunction in a patient. Preferred salts ofthe invention are labeled with one or more radioisotopes, preferablyincluding ¹¹C, ¹⁸F, ⁷⁶Br, or ¹²³I and more preferably ¹⁸F ⁷⁶Br, or ¹²³I.The invention provides a phosphonium cation tracer labeled with¹¹C-methyl group, e.g., [¹¹C]triphenylmethyl phosphonium (TPMP).Although suitable for use in medical centers situated at or near acyclotron, the short half-life time of ¹¹C, e.g., about 20 minutes,limits the use of [¹¹C]TPMP at distant medical centers. Preferred saltsof the invention comprise a ¹⁸F, ⁷⁶Br, ¹²³I, or a combination thereofand are suitable for use in peripheral medical facilities and PETclinics.

The present invention provides lipophilic salts comprising a cation ofFormula I or a subformula thereof which are preferentially taken up bydysfunctional mitochondria, e.g., mitochondria with suppressed orenhanced activity, and are suitable for use in imaging orradiotherapeutic applications. The invention provides imaging agentscomprising a radiolabeled labeled lipophilic cations, particularlylipophilic phosphonium or ammonium salts of the invention which has oneor more radioisotopes which is capable of binding to dysfunctionalmitochondria, e.g., mitochondria with suppressed or enhanced activity.More particularly, the radiolabeled labeled lipophilic phosphonium orammonium salts of the invention are suitable for use in measuringmitochondrial membrane potential (ΔΨm) in vivo under a variety ofconditions wherein the radiation emitted by the radioisotope of thelipophilic phosphonium or ammonium salt is utilized to form the image.In preferred embodiments, radiolabeled lipophilic phosphonium orammonium salts of the invention comprise one or more radioisotopescapable of emitting positron radiation and are suitable for use inpositron emission tomography (PET).

According to yet another aspect, the present invention providespharmaceutical compositions comprising radiolabeled labeled salts ofFormula I or the pharmaceutically acceptable salts or solvates thereof,which compositions are useful for the imaging variations inmitochondrial surface potential (ΔΨm), cells or tissues havingdysfunctional mitochondria, and diseases or disorders associated withdysfunctional mitochondria. The invention further provides methods ofimaging patients suffering from any of the above-recited diseases ordisorders with an effective amount of a salt or composition of theinvention.

Additionally this invention relates to the use of the salts of theinvention (particularly labeled salts of this invention emitting highenergy radiation) as therapeutic agents for the treatment of diseasesand disorders associated with dysfunctional mitochondria for which thelipophilic phosphonium or ammonium salts of the invention have highaffinity, e.g., disorders or diseases associated with dysfunctionalmitochondria activity. Typical disease and disorders include cancer,cardiovascular and liver diseases, degenerative disorders, autoimmunediseases and disorder, aging, DNA mutations, oxidative stress disorders,various myopaties, HIV, AIDS, and the like.

Preferred lipophilic cations, including phosphonium or ammonium salts,of the invention preferentially localize to cells possessingmitochondria with elevated or suppressed levels of activity, e.g.,dysfunctional mitochondrial activity.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1 through 19 show results of Examples 9 through 19 which follow.

DETAILED DESCRIPTION OF THE INVENTION

In addition to salts of Formula I, described above, the invention isfurther directed to lipophilic salts of Formula I (shown above) whereinthe compounds provided by the invention are lipophilic salts of FormulaI wherein

Ar is optionally substituted aryl having from 6 to 18 carbon atoms andbetween 1 and 3 rings, optionally substituted heteroaryl having from 3to about 18 carbon atoms, between 1 and about 3 rings and between 1 andabout 4 ring heteroatoms selected from N, O, and S, and optionallysubstituted aralkyl having between 7 and about 12 carbon atoms; and

R is optionally substituted C₁₋₆alkyl, optionally substitutedC₂₋₆alkenyl, optionally substituted C₂₋₆alkynyl, optionally substitutedC₁₋₆haloalkyl having at least one F, Cl, Br, or I atom, optionallysubstituted cycloalkyl having between 3 and about 8 ring carbon atoms,optionally substituted aralkyl having between 7 and about 12 carbonatoms, wherein at least one occurrence of R comprises at least oneradioisotope.

Preferred salts of the invention include salts having at least onephophonium cation of Formula I where E is phosphorus. Other preferredsalts of the invention include those having at least one ammonium cationof Formula I where E is nitrogen. Other preferred salts comprise amixture of cations according to Formula I where each cation may be aphophonium or ammonium cation.

Preferred salts of the invention comprise at least one R substitutentwhich comprises a radioisotope capable of emitting positrons. Typicallypreferred positron emitting radioisotopes suitable for use in Rsubstitutents include ¹¹C, ¹⁸F, ¹²³I or any combination thereof.

Other preferred salts provided by the invention include salts comprisinga cation of Formula II:

wherein

E is phosphorus or nitrogen; and

Ar¹, Ar², and Ar³ are independently selected from the group consistingof optionally substituted aryl, optionally substituted heteroaryl, andoptionally substituted aralkyl; and

R¹ is optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted haloalkyl,optionally substituted cycloalkyl, optionally substituted aralkyl,wherein at least one occurrence of R comprises at least oneradioisotope.

More preferably, cations according to Formula II which are provided bythe invention include those, wherein

Ar¹, Ar², and Ar³ are independently selected from the group consistingof optionally substituted aryl having from 6 to 18 carbon atoms andbetween 1 and 3 rings, optionally substituted heteroaryl having from 3to about 18 carbon atoms, between 1 and about 3 rings and between 1 andabout 4 ring heteroatoms selected from N, O, and S, and optionallysubstituted aralkyl having between 7 and about 12 carbon atoms; and

R¹ is optionally substituted C₁₋₆alkyl, optionally substitutedC₂₋₆alkenyl, optionally substituted C₂₋₆alkynyl, optionally substitutedC₁₋₆haloalkyl having at least one F, Cl, Br, or I atom, optionallysubstituted cycloalkyl having between 3 and about 8 ring carbon atoms,optionally substituted aralkyl having between 7 and about 12 carbonatoms, wherein at least one occurrence of R comprises at least oneradioisotope.

Particularly preferred cations of the invention according to Formula IIcomprise a R¹ group which is selected from the group consisting of¹¹C-methyl, optionally substituted C₁₋₆alkyl, optionally substitutedC₇₋₁₂aralkyl, optionally substituted C₆₋₁₂aryl, each of which issubstituted with one or more ¹¹C-methyl, ¹¹C-methoxy, ¹⁸F, ⁷⁶Br, ¹²³I,¹²⁵I, ¹³¹I, or a combination thereof More preferably, R¹ is ¹¹C-methyl,C₂₋₆alkyl substituted with one or more ¹⁸F, or benzyl substituted withone or more ¹⁸F, ⁷⁶Br, or ¹²³I.

The invention also provides salts comprising at least one cationaccording to Formula I or Formula II wherein R or R¹ comprises one ormore radioisotopes suitable for use in radiation therapy.

The present invention further provides salts comprising a cation ofFormula I which is represented by Formula III:

wherein

E is phosphorus or nitrogen;

R¹ is optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted haloalkyl,optionally substituted cycloalkyl, optionally substituted aralkyl,wherein at least one occurrence of R comprises at least oneradioisotope;

R², R³, and R⁴ are independently selected at each occurrence of R², R³,and R⁴ from the group consisting of hydrogen, halogen, cyano, nitro,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted (cycloalkyl)alkyl, optionally substituted alkylthio,optionally substituted alkylsulfinyl, or optionally substitutedalkylsulfonyl, and optionally substituted mono or dialkylcarboxamide.

Preferred R¹ groups of Formula III include halo-C₂₋₆alkyl group or ahalobenzyl group and more preferably R¹ of Formula III is selected fromthe group consisting ω-fluoro-C₂₋₆alkyl, ω-iodo-C₂₋₆alkyl group, ortho,meta or para-fluorobenzyl group, or ortho, meta or para-iodobenzylgroup.

Other preferred salts of the invention having a cation according toFormula I include those salts which comprise a cation according toFormula IV:

wherein

E is phosphorus or nitrogen;

Ar¹, Ar², and Ar³ are independently selected from the group consistingof optionally substituted aryl, optionally substituted heteroaryl, andoptionally substituted aralkyl; and

R⁵ and R⁶ are independently selected at each occurrence of R⁵ and R⁶from the group consisting of hydrogen, halogen, hydroxy, amino,optionally substituted alkyl, optionally substituted haloalkyl, andoptionally substituted alkoxy;

X is ¹¹C-methyl or a radioisotope of fluorine or iodine; and

m is a number from about 2 to about 6.

Yet other preferred salts of the invention having a cation according toFormula I include those salts which comprise a cation according toFormula V:

wherein

E is phosphorus or nitrogen;

Ar¹, Ar², and Ar³ are independently selected from the group consistingof optionally substituted aryl, optionally substituted heteroaryl, andoptionally substituted aralkyl; and

X is ¹¹C-methyl or a radioisotope of fluorine or iodine; and

m is a number from about 1 to about 5.

Still other preferred salts of the invention having a cation accordingto Formula I include those salts which comprise a cation according toFormula VI:

wherein

E is phosphorus or nitrogen;

Z is chloro, fluoro, hydroxy, or methoxy;

n is a number from 1 to about 12;

p and q are independently selected numbers from zero to about 5;

R¹ is optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted haloalkyl,optionally substituted cycloalkyl, optionally substituted aralkyl,wherein at least one occurrence of R comprises at least oneradioisotope; and

R² and R³ are independently selected at each occurrence of R² and R³from the group consisting of hydrogen, halogen, cyano, nitro, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted (cycloalkyl)alkyl, optionally substituted alkylthio,optionally substituted alkylsulfinyl, or optionally substitutedalkylsulfonyl, and optionally substituted mono or dialkylcarboxamide.

Other preferred salts of the invention having a cation according toFormula I include those salts which comprise a cation according toFormula VII

wherein

E is phosphorus or nitrogen;

p, q, and r are independently selected numbers from zero to about 4;

R¹ is optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted haloalkyl,optionally substituted cycloalkyl, optionally substituted aralkyl,wherein at least one occurrence of R comprises at least oneradioisotope;

R², R³, and R⁴ are independently selected at each occurrence of R², R³,and R⁴ from the group consisting of hydrogen, halogen, cyano, nitro,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted (cycloalkyl)alkyl, optionally substituted alkylthio,optionally substituted alkylsulfinyl, or optionally substitutedalkylsulfonyl, and optionally substituted mono or dialkylcarboxamide.

Particularly preferred salts comprising at least one pharmaceuticallyacceptable anion and at least one cation according to Formula I includethose salts comprising at least one cation selected from the groupconsisting of:

¹¹C-methyl-triphenylphosphonium ion;

¹¹C-methyl-tri-ortho-tolylphosphonium ion;

¹¹C-methyl-tri-meta-tolylphosphonium ion;

¹¹C-methyl-tri-para-tolylphosphonium ion;

¹⁸F-2-fluoroethyl-triphenylphosphonium ion;

¹⁸F-2-fluoroethyl-tri-ortho-tolylphosphonium ion;

¹⁸F-2-fluoroethyl-tri-meta-tolylphosphonium ion;

¹⁸F-2-fluoroethyl-tri-para-tolylphosphonium ion;

¹⁸F-3-fluoropropyl-triphenylphosphonium ion;

¹⁸F-3-fluoropropyl-tri-ortho-tolylphosphonium ion;

¹⁸F-3-fluoropropyl-tri-meta-tolylphosphonium ion;

¹⁸F-3-fluoropropyl-tri-para-tolylphosphonium ion;

¹⁸F-4-fluorobutyl-triphenylphosphonium ion;

¹⁸F-4-fluorobutyl-tri-ortho-tolylphosphonium ion;

¹⁸F-4-fluorobutyl-tri-meta-tolylphosphonium ion;

¹⁸F-4-fluorobutyl-tri-para-tolylphosphonium ion;

¹⁸F-2-fluorobenzyl-triphenylphosphonium ion;

¹⁸F-2-fluorobenzyl-tri-ortho-tolylphosphonium ion;

¹⁸F-2-fluorobenzyl-tri-meta-tolylphosphonium ion;

¹⁸F-2-fluorobenzyl-tri-para-tolylphosphonium ion;

¹⁸F-3-fluorobenzyl-triphenylphosphonium ion;

¹⁸F-3-fluorobenzyl-tri-ortho-tolylphosphonium ion;

¹⁸F-3-fluorobenzyl-tri-meta-tolylphosphonium ion;

¹⁸F-3-fluorobenzyl-tri-para-tolylphosphonium ion;

¹⁸F-4-fluorobenzyl-triphenylphosphonium ion;

¹⁸F-4-fluorobenzyl-tri-ortho-tolylphosphonium ion;

¹⁸F-4-fluorobenzyl-tri-meta-tolylphosphonium ion;

¹⁸F-4-fluorobenzyl-tri-para-tolylphosphonium ion;

¹⁸F-3-fluoro-4-formyl-benzyl-triphenylphosphonium ion;

¹⁸F-3-fluoro-4-formyl-benzyl-tri-ortho-tolylphosphonium ion;

¹⁸F-3-fluoro-4-formyl-benzyl-tri-meta-tolylphosphonium ion;

¹⁸F-3-fluoro-4-formyl-benzyl-tri-para-tolylphosphonium ion;

(¹⁸F-4-fluorobenzyl)-(2-chloroethyl)-diphenylphosphonium ion;

(¹⁸F-4-fluorobenzyl)-(3-chloropropyl)-diphenylphosphonium ion;

(¹⁸F-4-fluorobenzyl)-(4-chlorobutyl)-diphenylphosphonium ion;

(¹⁸F-4-fluorobenzyl)-(6-chloropentyl)-diphenylphosphonium ion;

(¹⁸F-4-fluorobenzyl)-(5-chlorohexyl)-diphenylphosphonium ion;

¹⁸F-2-fluoroethyl-tri(4-pyridyl)phosphonium ion;

¹⁸F-3-fluoropropyl-tri(4-pyridyl)phosphonium ion;

¹⁸F-4-fluorobutyl-tri(4-pyridyl)phosphonium ion;

¹⁸F-2-fluorobenzyl-tri(4-pyridyl)phosphonium ion;

¹⁸F-3-fluorobenzyl-tri(4-pyridyl)phosphonium ion;

¹⁸F-4-fluorobenzyl-tri(4-pyridyl)phosphonium ion; and

¹⁸F-3-fluoro-4-formyl-benzyl-tri(4-pyridyl)phosphonium ion.

Preferred radiolabeled salts of the invention including those saltscomprising a cation according to any one of Formula I, II, III, IV, V,VI, or VII, selectively localize to the mitochondria such that the ratioof radiation emitted from radiolabeled salts present in mitochondria tobackground radiation, e.g., radiolabeled salt not taken up inmitochondria is at least about 5:1. More preferably, salts of theinvention are selectively taken up in dysfunctional mitochondria suchthat the ratio of to normal mitochondria is at least about 5:1.

The salts of the invention, particularly the lipophilic salts of theinvention, have a distribution profile in the body which is a functionof mitochondrial integrity and are suitable for use as diagnostic toolsin the identification and imaging of various diseases and disordersassociated with mitochondrial dysfunction. Moreover, the salts of theinvention are useful diagnostic tools for assessing the efficacy ofexisting therapeutically drugs as well as the development of noveldrugs. For example, the effectiveness of drugs that trigger apoptosis(e.g., anticancer drugs) or suppress apoptosis (e.g., drugs that blockthe degenerative process in HIV) can be assessed by determining if theadministration of said drugs can be monitored by observing the change(effective) or effective) to ΔΨm by measuring ΔΨm using the salts andimaging methods of the invention.

Preferred compounds of the invention, particularly compounds suitablefor use in the imaging methods provided by the invention, include one ormore radioisotopes capable of emitting one or more forms of radiationwhich are suitable for detection with any standard radiology equipmentsuch as PET, SPECT, gamma cameras, MRI and the like. Preferredradioisotopes include tritium and isotopes of carbon, fluorine,technetium, iodine and other isotopes capable of emitting positrons.Particularly preferred radioisotopes include ¹¹C, ¹⁸F, ⁷⁶Br, and ¹²³I.

The present invention further provides method of imaging which comprisethe steps of:

providing at least one radiolabeled salt comprising a pharmaceuticallyacceptable anion and at least one cation according to any one of FormulaI, II, III, IV, V, VI, or VII;

contacting cells or tissues with the radiolabeled salt; and

making a radiographic image.

The imaging methods provided by the invention are suitable for assessingmitochondrial membrane potential (ΔΨm). More particularly, the imagingmethods of the present invention are suitable for measuring change inmitochondrial membrane potential over time to assess the efficacy oftherapeutic protocols or pharmaceutical treatments. Cells which exhibitsuppressed or enhanced rates of apoptosis frequently also exhibitdecreased or increased mitochondria activity. The salts provided by thepresent invention typically localize to cells in a concentrationproportional to the level of mitochondria activity. Thus frequently whencells are experiencing reduced levels of apoptosis (e.g., cancer cells),a greater portion of the salt of the invention administered to thepatient localizes to those cells, and vice versa, cells with enhancedlevels of apoptosis (e.g., auto immune disorders, tumor cells responsiveto chemotherapy agents) will accumulate less salt of the invention thannormal cells. Thus the imaging methods of the present invention aresuitable for use in imaging of cells, tissues or other physiologicaltargets which are experiencing suppressed or enhanced apoptosis.

The imaging methods of the present invention are generally suitable forimaging of any disease, disorder, or pathology which is related tomitochondria. Preferred diseases and disorders which are suitable forimaging include cancer (including neoplasms), cardiovascular diseases(including infraction and perfusion), liver diseases, degenerativediseases or disorders, autoimmune disorders, aging, HIV infections,myopathies caused by oxidative stress or DNA mutation, or diseases anddisorders associated with mitochardial dysfunction.

The imaging methods of the invention are also suitable for use inassessing efficacy of therapeutic drugs capable of triggering orsuppressing apoptosis. The imaging methods of the invention may also beused to assess the efficacy of chemotherapy or radiation treatmentprotocols used to retard or destroy cancer and other malignant tumors.

The imaging methods of the invention which are suitable for assessingthe efficacy of a therapeutic drug are also suitable in developing newtherapeutic agents which are capable of disrupting mitochondrialfunction in target tissue.

The radiolabeled lipophilic salts of the invention and imaging methodsusing same provide a non-invasive approach for early and sensitiveassessment of treatment efficacy within a few days of starting atherapeutic protocol compared to current assessment methods which mayrequire months. Most major anticancer drugs (e.g., taxol, cisplatin,vinblastine, and etoposide) induce their apoptotic effect via a cascadeof events in which the collapse of ΔΨm constitutes an early, obligatoryand irreversible step of the apoptotic process. Radiolabeled lipophilicsalts of the invention accumulate mainly in the mitochondria and indirect correlation with ΔΨm. Cells affected by the treatment willaccumulate radiolabeled lipophilic salts of the invention much less thannon-affected cells. Therefore, significant change between pre- andpost-treatment scan will indicate tumor responding to treatment and lackof differences will indicate non-responding tumors. Collapse of ΔΨmoccurs within hours after treatment with most therapeutic agents.

The ability to monitor the first event of the irreversible phase of theapoptotic process affords a noninvasive method for early and sensitivedetection of tumor response to treatment. In the clinical setting, theimaging methods provided by the present invention offer a powerful toolfor tailoring of chemotherapy strategies that will most benefit thepatient with reduced morbidity.

The radiolabeled lipophilic salts of the invention are also suitable foruse in developing these new generations of chemotherapy agents. Theradiolabeled lipophilic salts of the invention and imaging methods ofusing the same are suitable for use as non-invasive technique for anearly and sensitive assessment at the molecular level of treatmentefficacy in clinical studies. Moreover the imaging methods of theinvention are suitable for use in selecting suitable malignant targetsin test subjects, based on the functional integrity of mitochondria,upon which the novel drug can be tested.

The present invention further provides imaging methods suitable for usein the imaging of tumors with one or more salts having a cationaccording to Formula I or a subformula thereof. In preferred tumorimaging methods of the invention, the radiolabeled salt administered toa patient preferentially accumulates in mitochondria of malignant cellssuch that the concentration of radiolabeled cation of Formula I isgreater in the mitochondria of the malignant cell than the concentrationof the cation in adjacent normal cells.

The extent of cancerous disease (stage) is a major prognostic factor andnon-invasive staging using imaging technologies has a key role in designof treatment strategies (e.g., surgery vs. radio-chemotherapy vs.adjuvant chemotherapy). The lipophilic salts of the present inventionincluding salts having a cation according to Formula I accumulate inmalignant cells to a substantially greater extent than in normal cells.Administration of a radiolabeled salt of the present invention issuitable for the identification and imaging of malignant cells andtumors and is further suitable for measuring the stage of tumordevelopment.

The tumor imaging methods of the invention are particularly suitable incertain embodiments for imaging of cancers, more particularly forimaging neoplasms including a variety of lung, breast, and prostatecancers. Moreover, the tumor imaging method of the invention may be usedis capable of determining the extent of the cancerous disease (cancerstage).

The present invention provides methods of differentiating betweenmalignant tumors, such as neoplasms, and tissue suffering from a varietyof inflammation processes. The lipophilic salts of the inventionincluding salts having a cation of Formula I accumulate in malignantcells to a greater extent than in normal cells and accumulate incellular components of inflammatory processes to a lesser extent than innormal cells such that a differential distinction can be made betweenmalignant cells, normal cells and cells suffering from inflammation.Differential detection of malignancy will obviate the numerousunnecessary surgeries conducted each year and improve thecost-effectiveness of care management in oncology.

The tumor imaging methods of the present invention are capable ofdistinguishing between tissue suffering from an inflammatory process andmalignant lesions. While not wishing to be bound by theory, differentialdetection of malignant cells is possible because malignant cells have agreater accumulation of the salts of the invention than normal cells andtissues or cellular components of inflammatory processes typicallyaccumulate a lower concentration of the salts of the invention. Thus theconcentration of any salt of the invention in the malignant lesion issignificantly greater than normal tissue and tissue suffering from aninflammatory process.

The present invention further provides methods of imaging cardiovasculardiseases, particularly methods of imaging the myocardia Thecardiovascular imaging methods of the invention comprise theadministration of at least one compound according to Formula I, or asubformula thereof to a patient suffering from or susceptible to acardiovascular disease.

[18F]phosphonium cations of the invention are suitable for variouscardiovascular diseases, particularly myocardial imaging. Myocytescontain the highest concentration of mitochondria and therefore theheart is by far the major organ target of phosphonium cations. Inaddition, phosphonium cation maintain excellent perfusioncharacteristics permitting high-contrast imaging of the heart Infarctand heart failure involve apoptosis followed by necrosis processes.[¹⁸F]Phosphonium cations are capable to accurately distinguish betweenmyocardial segments in which the apoptotic process cannot be stopped bymedication and revascularization, and myocardial area that can besalvaged by intervention.

Preferred imaging methods provided by the invention include the use oflipophilic salts according to any one of Formula I, II, III, IV, V, VI,or VII which are capable of generating at least a 2:1 target tobackground ratio of radiation intensity, or more preferably about a 5:1,about a 10:1 or about a 15:1 ratio of radiation intensity between targetand background. In certain preferred methods the radiation intensity ofthe target tissue is more intense than that of the background. In otherembodiments, the invention provides methods where the radiationintensity of the target tissue is less intense than that of thebackground. Generally, any difference in radiation intensity between thetarget tissue and the background which is sufficient to allow foridentification and visualization of the target tissue is sufficient foruse in the methods of the present invention.

In preferred methods of the invention the compounds of the invention areexcreted from tissues of the body quickly to prevent prolonged exposureto the radiation of the radiolabeled compound administered to thepatient. Typically compounds according to Formula I or any subformulathereof are eliminated from the body in less than about 24 hours. Morepreferably, compounds of the invention are eliminated from the body inless than about 16 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours,90 minutes, or 60 minutes. Typically preferred compounds are eliminatedin between about 60 minutes and about 120 minutes.

Preferred compounds of the invention are stable in vivo such thatsubstantially all, e.g., more than about 50%, 60%, 70%, 80%, or morepreferably 90% of the injected compound is not metabolized by the bodyprior to excretion.

Compounds and salts of the invention and imaging methods of theinvention are useful in imaging a variety of conditions includingcancer, cardiovascular and liver diseases, HIV, AIDS, autoimmunedisease, degenerative disorders, neoplasms, and the like.

Typical subjects to which compounds of the invention may be administeredwill be mammals, particularly primates, especially humans. Forveterinary applications, a wide variety of subjects will be suitable,e.g. livestock such as cattle, sheep, goats, cows, swine and the like;poultry such as chickens, ducks, geese, turkeys, and the like; anddomesticated animals particularly pets such as dogs and cats. Fordiagnostic or research applications, a wide variety of mammals will besuitable subjects including rodents (e.g. mice, rats, hamsters),rabbits, primates, and swine such as inbred pigs and the like.Additionally, for if vitro applications, such as in vitro diagnostic andresearch applications, body fluids and cell samples of the abovesubjects will be suitable for use such as mammalian, particularlyprimate such as human, blood, urine or tissue samples, or blood urine ortissue samples of the animals mentioned for veterinary applications.

The present invention also provide packaged pharmaceutical compositionscomprising a pharmaceutical acceptable carrier and a salt comprising atleast one pharmaceutically acceptable anion and a cation according toany one of Formula I, II, III, IV, V, VI, or VII. In certain embodimentsthe packaged pharmaceutical composition will comprise the reactionprecursors necessary generate the compound or salt according to FormulaI or subformula thereof upon combination with a radiolabeled precursor.Other packaged pharmaceutical compositions provided by the presentinvention further comprise indicia comprising at least one of:

instructions for using the composition to image cells or tissues havingincreased or suppressed mitochondrial activity, or

instructions for using the composition to assess therapeutic effect of adrug protocol administered to a patient, or

instructions for using the composition to selectively image malignantcells and tumors in the presence of inflammation, or

instructions for using the composition to measure mitochondrial membranepotential (ΔΨm).

In certain preferred embodiments, the invention provides a kit accordingto the invention contains from about 1 to about 30 mCi of theradionuclide-labeled imaging agent described above, in combination witha pharmaceutically acceptable carrier. The imaging agent and carrier maybe provided in solution or in lyophilized form. When the imaging agentand carrier of the kit are in lyophilized form, the kit may optionallycontain a sterile and physiologically acceptable reconstitution mediumsuch as water, saline, buffered saline, and the like.

The present invention further provides apparatus and synthetic protocolsfor the automated synthesis of ¹¹C, ¹⁸F, ⁷⁶Br, or ¹²³I labeled salts ofthe invention, including salts comprising a cation according to any oneof Formula I, II, III, VI, V, VII, and VII, and preparation ofpharmaceutical compositions comprising same. The half-life (120 min) ofF-18 allows for distribution of cationic probes from central cyclotronto satellite PET scanners, similarly to the rapidly evolvingdistribution system adopted for [18F]FDG. Tagging the cationic probeswith I-123 will allow for distribution from a manufacturing center tomedical institutions equipped with SPECT.

Imaging agents of the invention may be used in accordance with themethods of the invention by one of skill in the art, e.g., byspecialists in nuclear medicine, to image sites having a dysfunctionalmitochondria, e.g., mitochondria exhibiting aberrant activity, in asubject or patient. Any site having a dysfunctional mitochondria, e.g.,mitochondria exhibiting aberrant activity, may be imaged by the imagingmethods and imaging agents of the present invention.

Images can be generated by virtue of differences in the spatialdistribution of the imaging agents which accumulate at a site having adysfunctional mitochondria, e.g., mitochondria exhibiting aberrantactivity. The spatial distribution may be measured using any meanssuitable for the particular label, for example, a gamma camera, a PETapparatus, a SPECT apparatus, and the like. The extent of accumulationof the imaging agent may be quantified using known methods forquantifying radioactive emissions. A particularly useful imagingapproach employs more than one imaging agent to perform simultaneousstudies. Alternatively, the imaging method may be carried out aplurality of times with increasing administered dose of the saltaccording to Formula I to perform successive studies using thesplit-dose image subtraction method.

Preferably, a detectably effective amount of the imaging agent of theinvention is administered to a subject. In accordance with theinvention, “a detectably effective amount” of the imaging agent of theinvention is defined as an amount sufficient to yield an acceptableimage using equipment which is available for clinical use. A detectablyeffective amount of the imaging agent of the invention may beadministered in more than one injection. The detectably effective amountof the imaging agent of the invention can vary according to factors suchas the degree of susceptibility of the individual, the age, sex, andweight of the individual, idiosyncratic responses of the individual, thedosimetry. Detectably effective amounts of the imaging agent of theinvention can also vary according to instrument and film-relatedfactors. Optimization of such factors is well within the level of skillin the art.

The amount of imaging agent used for diagnostic purposes and theduration of the imaging study will depend upon the radionuclide used tolabel the agent, the body mass of the patient, the nature and severityof the condition being treated, the nature of therapeutic treatmentswhich the patient has undergone, and on the idiosyncratic responses ofthe patient. Ultimately, the attending physician will decide the amountof imaging agent to administer to each individual patient and theduration of the imaging study.

The compounds herein described may have one or more asymmetric centersor planes. Compounds of the present invention containing anasymmetrically substituted atom may be isolated in optically active orracemic forms. It is well known in the art how to prepare opticallyactive forms, such as by resolution of racemic forms (racemates), byasymmetric synthesis, or by synthesis from optically active startingmaterials. Resolution of the racemates can be accomplished, for example,by conventional methods such as crystallization in the presence of aresolving agent, or chromatography, using, for example a chiral HPLCcolumn. Many geometric isomers of olefins, C═N double bonds, and thelike can also be present in the compounds described herein, and all suchstable isomers are contemplated in the present invention. Cis and transgeometric isomers of the compounds of the present invention aredescribed and may be isolated as a mixture of isomers or as separatedisomeric forms. All chiral (enantiomeric and diastereomeric), andracemic forms, as well as all geometric isomeric forms of a structureare intended, unless the specific stereochemistry or isomeric form isspecifically indicated.

When any variable occurs more than one time in any constituent orformula for a compound, its definition at each occurrence is independentof its definition at every other occurrence. Thus, for example, if agroup is shown to be substituted with 0-2 R*, then said group mayoptionally be substituted with up to two R* groups and R* at eachoccurrence is selected independently from the definition of R*. Also,combinations of substituents and/or variables are permissible only ifsuch combinations result in stable compounds.

As indicated above, various substituents of the various formulae(compounds of Formula I, II, III, IV, V, VI, or VII) are “optionallysubstituted”, including Ar, Ar¹, Ar², Ar³, R, R¹, R², R³, R⁴, R⁵, R⁶, X,X¹, X², X³, X⁴, and Z of Formula I, II, III, IV, V, VI and VII, and suchsubstituents as recited in the sub-formulae such as Formula I andsubformulae. The term “substituted,” as used herein, means that any oneor more hydrogens on the designated atom or group is replaced with aselection from the indicated group of substituents, provided that thedesignated atom's normal valence is not exceeded, and that thesubstitution results in a stable compound. When a substituent is oxo(keto, i.e., ═O), then 2 hydrogens on an atom are replaced. The presentinvention is intended to include all isotopes (including radioisotopes)of atoms occurring in the present compounds.

When substituents such as Ar, Ar¹, Ar², Ar³, R, R¹, R², R³, R⁴, R⁵, R⁶,X, X¹, X², X³, X⁴, and Z of Formula I and subformulae thereof, and suchsubstituents as recited in the sub-formulae are further substituted,they may be so substituted at one or more available positions, typically1 to 3 or 4 positions, by one or more suitable groups such as thosedisclosed, herein. Suitable groups that may be present on a“substituted” R¹, R², R or other group include e.g., halogen; cyano;hydroxyl; nitro; azido; alkanoyl (such as a C₁₋₆ alkanoyl group such asacyl or the like); carboxamido; alkyl groups (including cycloalkylgroups, having 1 to about 8 carbon atoms, preferably 1, 2, 3, 4, 5, or 6carbon atoms); alkenyl and alkynyl groups (including groups having oneor more unsaturated linkages and from 2 to about 8, preferably 2, 3, 4,5 or 6, carbon atoms); alkoxy groups having one or more oxygen linkagesand from 1 to about 8, preferably 1, 2, 3, 4, 5 or 6 carbon atoms;aryloxy such as phenoxy; alkylthio groups including those having one ormore thioether linkages and from 1 to about 8 carbon atoms, preferably1, 2, 3, 4, 5 or 6 carbon atoms; alkylsulfinyl groups including thosehaving one or more sulfinyl linkages and from 1 to about 8 carbon atoms,preferably 1, 2, 3, 4, 5, or 6 carbon atoms; alkylsulfonyl groupsincluding those having one or more sulfonyl linkages and from 1 to about8 carbon atoms, preferably 1, 2, 3, 4, 5, or 6 carbon atoms; aminoalkylgroups including groups having one or more N atoms and from 1 to about8, preferably 1, 2, 3, 4, 5 or 6, carbon atoms; carbocyclic aryl having6 or more carbons and one or more rings, (e.g., phenyl, biphenyl,naphthyl, or the like, each ring either substituted or unsubstitutedaromatic); arylalkyl having 1 to 3 separate or fused rings and from 6 toabout 18 ring carbon atoms, with benzyl being a preferred arylalkylgroup; arylalkoxy having 1 to 3 separate or fused rings and from 6 toabout 18 ring carbon atoms, with O-benzyl being a preferred arylalkoxygroup; or a saturated, unsaturated, or aromatic heterocyclic grouphaving 1 to 3 separate or fused rings with 3 to about 8 members per ringand one or more N, O or S atoms, e.g. coumarinyl, quinolinyl,isoquinolinyl, quinazolinyl, pyridyl, pyrazinyl, pyrimidyl, furanyl,pyrrolyl, thienyl, thiazolyl, triazinyl, oxazolyl, isoxazolyl,imidazolyl, indolyl, benzofuranyl, benzothiazolyl, tetrahydrofuranyl,tetrahydropyranyl, piperidinyl, morpholinyl, piperazinyl, andpyrrolidinyl. Such heterocyclic groups may be further substituted, e.g.with hydroxy, alkyl, alkoxy, halogen and amino.

As used herein, “alkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups, having thespecified number of carbon atoms. Examples of alkyl include, but are notlimited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl,t-butyl, n-pentyl, and s-pentyl. Preferred alkyl groups are C₁₋₆ alkylgroups. Especially preferred alkyl groups are methyl, ethyl, propyl,butyl, and 3-pentyl. The term C₁₋₄ alkyl as used herein includes alkylgroups consisting of 1 to 4 carbon atoms, which may contain acyclopropyl moiety. Suitable examples are methyl, ethyl, andcyclopropylmethyl.

“Cycloalkyl” is intended to include saturated ring groups, having thespecified number of carbon atoms, such as cyclopropyl, cyclobutyl,cyclopentyl, or cyclohexyl. Cycloalkyl groups typically will have 3 toabout 8 ring members.

In the term “(C₃₋₈ cycloalkyl)C₁₋₄ alkyl”, cycloalkyl, and alkyl are asdefined above, and the point of attachment is on the alkyl group. Thisterm encompasses, but is not limited to, cyclopropylmethyl,cyclohexylmethyl, and cyclohexylmethyl.

“Alkenyl” is intended to include hydrocarbon chains of either a straightor branched configuration comprising one or more unsaturatedcarbon-carbon bonds, which may occur in any stable point along thechain, such as ethenyl and propenyl. Alkenyl groups typically will have2 to about 8 carbon atoms, more typically 2 to about 6 carbon atoms.

“Alkynyl” is intended to include hydrocarbon chains of either a straightor branched configuration comprising one or more carbon-carbon triplebonds, which may occur in any stable point along the chain, such asethynyl and propynyl. Alkynyl groups typically will have 2 to about 8carbon atoms, more typically 2 to about 6 carbon atoms.

“Haloalkyl” is intended to include both branched and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms, substituted with 1 or more halogen atoms. Examples ofhaloalkyl include, but are not limited to, mono-, di-, ortri-fluoromethyl, mono-, di-, or tri-chloromethyl, mono-, di-, tri-,tetra-, or penta-fluoroethyl, and mono-, di-, tri-, tetra-, orpenta-chloroethyl. Typical haloalkyl groups will have 1 to about 8carbon atoms, more typically 1 to about 6 carbon atoms.

“Alkoxy” represents an alkyl group as defined above with the indicatednumber of carbon atoms attached through an oxygen bridge. Examples ofalkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy,i-propoxy, n-butoxy, 2-butoxy, t-butoxy, n-pentoxy, 2-pentoxy,3-pentoxy, isopentoxy, neopentoxy, n-hexoxy, 2-hexoxy, 3-hexoxy, and3-methylpentoxy. Alkoxy groups typically have 1 to about 8 carbon atoms,more typically 1 to about 6 carbon atoms.

“Halolkoxy” represents a haloalkyl group as defined above with theindicated number of carbon atoms attached through an oxygen bridge.

As used herein, the term “alkylthio” includes those groups having one ormore thioether linkages and preferably from 1 to about 8 carbon atoms,more typically 1 to about 6 carbon atoms.

As used herein, the term “alkylsulfinyl” includes those groups havingone or more sulfoxide (SO) linkage groups and typically from 1 to about8 carbon atoms, more typically 1 to about 6 carbon atoms.

As used herein, the term “alkylsulfonyl” includes those groups havingone or more sulfonyl (SO₂) linkage groups and typically from 1 to about8 carbon atoms, more, typically 1 to about 6 carbon atoms.

As used herein, the term “alkylamino” includes those groups having oneor more primary, secondary and/or tertiary amine groups and typicallyfrom 1 to about 8 carbon atoms, more typically 1 to about 6 carbonatoms.

“Halo” or “halogen” as used herein refers to fluoro, chloro, bromo, oriodo; and “counter-ion” is used to represent a small, negatively chargedspecies such as chloride, bromide, hydroxide, acetate, sulfate, and thelike.

As used herein, “carbocyclic group” is intended to mean any stable 3- to7-membered monocyclic or bicyclic or 7- to 13-membered bicyclic ortricyclic group, any of which may be saturated, partially unsaturated,or aromatic. In addition to those exemplified elsewhere herein, examplesof such carbocycles include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclooctyl,[3.3.0]bicyclooctanyl, [4.3.0]bicyclononanyl, [4.4.0]bicyclodecanyl,[2.2.2]bicyclooctanyl, fluorenyl, phenyl, naphthyl, indanyl, andtetrahydronaphthyl.

As used herein, the term “heterocyclic group” is intended to includesaturated, partially unsaturated, or unsaturated (aromatic) groupshaving 1 to 3 (preferably fused) rings with 3 to about 8 members perring at least one ring containing an atom selected from N, O or S. Thenitrogen and sulfur heteroatoms may optionally be oxidized. The term or“heterocycloalkyl” is used to refer to saturated heterocyclic groups.

The heterocyclic ring may be attached to its pendant group at anyheteroatom or carbon atom that results in a stable structure. Theheterocyclic rings described herein may be substituted on carbon or on anitrogen atom if the resulting compound is stable. A nitrogen in theheterocycle may optionally be quaternized. As used herein, the term“aromatic heterocyclic system” is intended to include any stable 5- to7-membered monocyclic or 10- to 14-membered bicyclic heterocyclicaromatic ring system which comprises carbon atoms and from 1 to 4heteroatoms independently selected from the group consisting of N, O andS. It is preferred that the total number of S and O atoms in thearomatic heterocycle is not more than 2, more preferably not more than1.

Examples of heterocycles include, but are not limited to, thoseexemplified elsewhere herein and further include acridinyl, azocinyl,benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl,benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl,benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl,NH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isobenzofuranyl, isochromanyl,isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl,isoxazolyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl,oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl; 1,2,5-oxadiazolyl,1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidinyl,phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl,phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl,1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, and xanthenyl.

Preferred heterocyclic groups include, but are not limited to,pyridinyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl,pyrrolidinyl, morpholinyl, piperidinyl, piperazinyl, and imidazolyl.Also included are fused ring and spiro compounds containing, forexample, the above heterocycles.

As used herein, the term “carbocyclic aryl” includes groups that contain1 to 3 separate or fused rings and from 6 to about 18 ring atoms,without hetero atoms as ring members. Specifically preferred carbocyclicaryl groups include phenyl, and naphthyl including 1-napthyl and2-naphthyl.

A “pharmaceutically acceptable carrier” refers to a biocompatiblesolution, having due regard to sterility, pH, isotonicity, stability,and the like and can include any and all solvents, diluents (includingsterile saline, Sodium Chloride Injection, Ringer's Injection, DextroseInjection, Dextrose and Sodium Chloride Injection, Lactated Ringer'sInjection and other aqueous buffer solutions), dispersion media,coatings, antibacterial and antifungal agents, isotonic agents, and thelike. The pharmaceutically acceptable carrier may also containstabilizers, preservatives, antioxidants, or other additives, which arewell known to one of skill in the art, or other vehicle as known in theart.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the disclosed compounds wherein the parent compound is modified bymaking non-toxic acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. The pharmaceutically acceptable salts include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids. Forexample, conventional non-toxic acid salts include those derived frominorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic,phosphoric, nitric and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, pamoic, malefic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicylic, mesylic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, HOOC—(CH₂)n—COOH where n is 0-4, and thelike. The pharmaceutically acceptable salts of the present invention canbe synthesized from a parent compound that contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting free acid forms of these compounds with astoichiometric amount of the appropriate base (such as Na, Ca, Mg, or Khydroxide, carbonate, bicarbonate, or the like), or by reacting freebase forms of these compounds with a stoichiometric amount of theappropriate acid. Such reactions are typically carried out in water orin an organic solvent, or in a mixture of the two. Generally,non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, oracetonitrile are preferred, where practicable. Lists of additionalsuitable salts may be found, e.g., in Remington's PharmaceuticalSciences, 17th ed., Mack Publishing Company, Easton, Pa., p. 1418(1985).

Because of the correlation between elevated or suppressed mitiochondiaactivity and a variety of diseases and disorders, an imaging agent andmethods of imaging using same that can assessing changes in mitochondriasurface potential is an effective diagnostic tool for testing for thepresence of a variety of disease states associated with triggering orsuppressing apoptosis in cells. Moreover, imaging agents suitable foruse in imaging or assessing changes in mitochondria surface potentialare suitable for use in studying a variety of diseases including cancer,cardiovascular or liver diseases, HIV, AIDS, autoimmune disease,degenerative disorders, neoplasms, and the like.

EXAMPLES

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way. The contents ofall cited references (including literature references, issued patents,published patent applications) as cited throughout this application arehereby expressly incorporated by reference. The practice of the presentinvention will employ, unless otherwise indicated, conventionaltechniques, which are within the skill of the art. Such techniques areexplained fully in the literature.

Example 1 [¹⁸F]3-fluoropropyltriphenyl-phosphonium ion ([¹⁸F]FPTP)

The synthesis starts with the [¹⁸F]fluoride from the cyclotron targettransferred onto an anion exchange column (Trap and Release column(DW-TRC) D and W, Inc., Oakdale, Tenn., USA). The column is eluted withaqueous potassium carbonate (2.3 mg dissolved in 0.3 mL) into a 5 ccv-vial containing Kryptofix. The Kryptofix, potassium carbonate,[¹⁸F]fluoride mixture is dried at 120 C; and, 7 mg of propyl ditosylate(Aldrich) is added in 0.5 mL acetonitrile. After heating at 80° C. for 5minutes, 21 mg of triphenylphosphine (Aldrich) in 0.5 mL of toluene isadded. The acetonitrile is evaporated away and the toluene mixtureheated to boiling for 3-5 minutes. After evaporating the toluene andcooling the vial, 0.5 mL of high-pressure liquid chromatography (HPLC)solvent [35:65 acetonitrile:water (0.1 M ammonium formate)] is added tothe vial. The mixture is filtered through a 0.45 μm Teflon HPLC filter(Alltech 13 mm) and injected onto a preparative HPLC column (WatersNovapak C-18 6 μm, 7.8×300 mm) at 7 ml/min for purification. The productis collected on a rotary evaporator modified to allow addition andremoval of solvents, the HPLC solvent evaporated and the radiolabeledphosphonium salt dissolved in sterile normal saline. The overall decaycorrected radiochemical yield of [18F]FPTP calculated from starting[18F]fluoride is 12 percent. After sterile filtration (PALL-Gelman 0.2μm Tuffryn) into a sterile vial, the solution is checked forradiochemical, chemical purity and specific activity by analytical HPLC[40:60 acetonitrile:water (0.1 M ammonium formate), Waters Novapak C-1860 Å4 μm, 3.9×150 mm] at 3 ml/min with a known concentration of coldstandard characterized solution of 3-fluoropropyltriphenyl-phosphoniumbromide [physical data: mp 313-316 C; ¹H NMR (CDCl₃, δ) 1.81-2.17 (m,2H), 4.01-4.11 (m, 2H), 4.72-4.75 (m, 1H), 4.87-4.90 (m, 1H), 7.69-7.88(m, 15H)]. The synthesis is summarized in Scheme 1.

Example 2 [¹⁸F]2-fluoroethyltriphenyl-phosphonium ion ([¹⁸F]FETP)

[¹⁸F]FETP was prepared according to the method used to prepare [¹⁸F]FPTPas described in Example 1 supra.

Example 3 [¹⁸F]2-fluorobutyltriphenyl-phosphonium ion ([¹⁸F]FBTP)

[¹⁸F]FBTP was prepared according to the method used to prepare [¹⁸F]FPTPas described in Example 1 supra.

Example 4 [¹⁸F]fluorobenzyltriphenyl-phosphonium ion

After collecting and drying the [¹⁸F]fluoride in the potassiumcarbonate/Kryptofix (as described in Example 1),trimethylammnoniumbenzaldehyde triflate salt (7 mg) in 0.2 mL ofdimethylsulfoxide(vacuum distilled from barium oxide prior to use) isadded to the mixture. After heating at 120° C. for 5 minutes, thealdehyde is diluted with 5 mL of HPLC water and collected on a C-18solid phase extraction cartridge (Waters C-18 Plus Sep-Pak) then washedwith 10 ml of HPLC water and dried by inert gas flow through thecartridge for 3 minutes. The aldehyde is eluted from the cartridge with2 mL of diethylether (Aldrich) and passed through 10% sodium borohydrideon basic alumina (Aldrich-200-400 mg) to reduce it to the alcohol. Thealcohol is subsequently converted to [¹⁸F]fluorobenzyl bromide by mixingwith triphenylphosphine dibromide (75-100 mg) in 1 mL of methylenechloride for 5 minutes. After passing through a silica solid phaseextraction cartridge (Waters Silica Classic Sep-Pak) and washing with 1mL of methylene chloride, the [¹⁸F]fluorinated benzyl bromide is addedto 21 mg of triphenylphosphine (or its analog) dissolved in 0.5 mLtoluene in a 5 ml v-vial. The methylene chloride/ether solvent isevaporated away at low heat with inert gas flow, the vial capped andheated to boiling for 3-5 minutes. After evaporating the toluene andcooling the vial, 0.5 mL of high-pressure liquid chromatography (HPLC)solvent [50:50 acetonitrile:water (0.1 M ammonium formate)] is added tothe vial. The mixture is filtered through a 0.45 μm Teflon HPLC filter(Alltech 13 mm) and injected onto a preparative HPLC column (WatersNovapak C-18 6 μm, 7.8×300 mm) at 7 ml/min for purification. The productis collected on a rotary evaporator modified to allow addition andremoval of solvents, the HPLC solvent evaporated and the radiolabeledphosphonium salt dissolved in sterile normal saline. After sterilefiltration (PALL-Gelman 0.2 μm Tuffryn) into a sterile vial, thesolution is checked for radiochemical, chemical purity and specificactivity by analytical HPLC [40:60 acetonitrile:water (0.1 M ammoniumformate), Waters Novapak C-18 60 Å4 μm, 3.9×150 mm] at 3 ml/min andcompared to a known standard of p-fluorobenzyltriphenylphosphonium ionas in example 1. The overall decay corrected radiochemical yield of[18F]fluorbenzyltriphenyl phosphonium ion calculated from starting[18F]fluoride is 14 percent. [Physical Data: mp 313-316 C; ¹H NMR(D⁶-dmso, δ) 5.17-5.21 (d, 2H), 6.99-7.08 (m, 4H), 7.67-7.94 (m, 15H)].The synthesis is summarized in Scheme 2.

Example 5 Radiolabeled diphenyl(haloalkyl)([¹⁸F]fluorobenzyl)phosphoniumion

Radiolabeled haloalkylphosphonium ion derivatives are prepared by thesynthesis of a ¹⁸F-fluorobenzyl halide as described in Example 4. Theradiolabeled benzyl halide will be attached to diphenylphosphine tocreate a radiolabeled phosphine: Next the radiolabeled phosphine will beconverted to the haloalkylphosphonium ion by reaction with theappropriate haloalkyl iodide. The synthesis of the radiolabeledchloroalkyl phosphonium ion is summarized in Scheme 3. Other haloalkylspecies including chloro, bromo and iodo attached to alkyl chains ofvarying length, branching and site of halide substitution can also beprepared.

Example 6 radiolabled [¹⁸F]fluoroalkyl-tripyridylphosphonium ion andradiolabled [¹⁸F]fluorobenzyl-tripyridylphosphonium ion

Radiolabeled phosphonium ions containing pyridyl rings are preparedaccording to the generalized reaction scheme provided in Scheme 4. Theradiosynthesis involves the reaction of ¹⁸F-fluoroalkyl (Example 1) andbenzyl (Example 4) moieties as described above with tripyridylphosphine.

Example 7 Radiolabeled Ammonium Ions

The procedures described in Examples 1-6 for preparation of radiolabeledphosphonium ions are also applicable for the preparation of quaternaryammonium ions as illustrated in Scheme 5. Quaternary ammonium ions havecomparable biodistribution to phosphonium ions.

Example 8 Whole Body PET/SPECT Imaging

One imaging protocol suitable for delivery of a salt of the inventioninvolves intravenous administration of the salt and acquisition ofstatic scan for several minutes per bed position. The exact scanduration may be varied depending upon patient size, salt dosage, and thenature of the tissue to be imaged. However imaging parameters forimaging primates, particularly humans, can be modified as necessary byone skilled in the radiological arts and familiar with PET and/or SPECTimaging with other radiopharmaceutical agents.

Example 9 Radiolabeling of F-18-FBnTP and Stability in Plasma AfterInjection of the Agent into Mice

Radiochemical purity of F-18-FBnTP and its stability in vivo wasmeasured by chromatography.

Methods:

Preparation of F-18-fluorobenzyl triphenyl phosphonium (F-18-FBnTP): Thesynthesis of labeling FBnTP with F-18 in the ortho position is describedschematically below. After collecting the F-18-fluoride in Kryptofix (aswith the fluoroalkyl derivatives), a nitrobenzaldehyde in acetonitrileis added to the mixture. After heating, the aldehyde is reduced to thealcohol and subsequently converted to a radiolabeled benzyl halide. Thefluorinated benzyl halide is reacted with triphenylphosphine or itsanalog in toluene. The mixture is purified and quality controlperformed, as discussed above

Samples of mouse plasma were obtained from heparinized wholebloodcollected 5, 15 and 30 min p.i. of the tracer. To eliminate bindingto plasma proteins, the plasma was added to solid urea to give a finalconcentration of 8M urea. The plasma-urea was loaded into a columnswitch HPLC system (Hilton 2000), in which the plasma passes through asmall capture column (Oasis Sorbent, Waters Corp.), which retainslipophilic solutes, while polar species fail to bind and are detected asthey pass through a positron detector. After four minutes, the capturecolumn is free of plasma proteins and polar species. Then, the contentsof the capture column was swept onto an analytical column (ProdigyODS-3, Phenomenex) by 40% acetonitrile, 60% triethylamine acetate bufferpH 4.1 at 1 mL/min, where separation of the parent compound andlipophilic metabolites occurs. The effluent from the analytical columnalso passes through the positron flow detector. The proportion of eachspecies is determined from the area under each chromatographic peak.Results: Radiochemical purity of the 18F-labeled FBnTP was more than95%. Chromatography of plasma revealed a single radio-peak, comprising97% of total activity with no other peak observed. FIG. 1 depicts thechromatogram of plasma collected 30 min p.i., and the parental compoundincubated for a similar time period (30 min) in buffered saline. Plasmaactivity and parental compound peaked at same time (˜6 min).

These radiochromatograms are good evidence of the F-18-FBnTP can belabeled with high radiochemical purity and stable more than 30 min afterintravenous injection of the tracer in mice in vivo.

Example 10 Stability of FBnTP Fluorination

Methods: Mice were injected via the tail vein with 25 μCi of F-18-PhC; 5and 30 min p.i., the left or right femur bone was removed, and boneradioactivity together with standard (1:100 of injected dose) werecounted in a gamma counter. A parallel group of mice was injected withfree fluorides (F-18). Three mice of each group were studied at eachtime point. Radioactivity is represented as percentage of injected dosed(% ID) and total bone uptake was calculated as activity in femur bone×20.

Results: FIG. 2 depicts the total bone uptake at 5 and 30 min p.i. Themarginal bone uptake of F-18-PhC, compared to the bone uptake in miceinjected with F-18 only, indicates the stability of fluorination of thephosphonium compound. The minimal bone uptake of F-18-FBnTP in boneindicate the lack of free fluorides, meaning the stability offluorination

Example 11 Mitochondria Membrane Potential (MMP)-Dependent Uptake

The extent of MMP-dependent cellular uptake of F-18-FBnTP was assessedusing CCCP, a known protonophore that selectively abolishes the MMP.

Methods: Human lung carcinoma H549 cells (10/ml) were incubated with 0.1μCi/ml F-18-FBnTP for 30 min. Samples of the suspension (1 mL) weretransferred to Eppendorf's vials and placed in a 370 C bath. Varyingconcentrations (30, 60, 90, 120 μM) of CCCP were added to thesuspension. After 30 min of incubation with CCCP, the Eppendorf vialswere centrifuged for 1 min, and activity in pellet and supernatant wasimmediately counted.

Results: FIG. 3 depicts the cellular uptake of F-18-FBnTP in thepresence of varying concentrations of CCCP. CCCP induced adose-dependent decrease of the F-18-FBnTP cellular uptake. The largemajority (86%) of F-18-FBnTP cellular uptake is MMP-dependent.

Example 12 Biodistribution of Novel F-18-fluorophosphoinium CationsComparison with Various Tracers

Methods: The biodistribution of novel phosphonium compoundsF-18-fluorbenzyl triphenyl phosphonium (FBnTP) and F-18-fluopropyltriphenyl phosphonium cation (FPTP) was studies in adult mice andcompared with the tracers C-11-triphenylmethyl phosphonium (TPMP),tetraphenyl phosphonium (TPP) and Tc-99m-sestamibii (MIBI). F-18-FBnTPwas prepared as describe above. Preparation of FPTP is described below.TPMP was prepared as previously described (Madar I, J Nucl Med. 1999;40:1180-5). TPP and MIBI were purchased from (NEN and Dupont,respectively). Three to five mice were used for the biodistributionstudy of each tracer. Nonanesthetized animals were injected i.v. withtracer solution (FBnTP or FPTP, TPMP 25 μCi; MIBI 40 μCi; TPP 2 μCi, allin a volume of 0.2 mL saline) then killed by neck dislocation at 60 minafter injection. The organs and tissues of interest were removed andcounted by a radioactivity counter along with standards (1:1000) (LKBWallac, 1282 Compugamma CS).

Preparation of F-18-fluoropropyl triphenyl phosphonium ion (FPTP)

Schematic drawing of F-18-FPTP synthesis is given below. F-18-FPTP wasfirst prepared as the nonradioactive compound, fluoropropyltriphenylphosphonium bromide, from 1-fluoro-3-bromopropane andtriphenylphosphine. The cold compound was characterized by proton andcarbon-13 NMR, as well as HPLC. This standard was used for comparisonduring the purification, quality control and determination of specificactivity of the radiolabeled F-18-FPTP. The F-18-FPTP was synthesized asdescribed in FIG. 3. Briefly, 1,3-ditosylpropane in acetonitrile wasadded to a dried vial containing F-18-fluoride, Kryptofix and potassiumcarbonate. After heating at 80° C. for 4 minutes on a heat gun,triphenylphosphine in toluene was added to the vial. After 5 minutes,the volume of the solution decreased to a few microliters. After coolingto room temperature, HPLC solvent was added to the vial. The mixture wasfiltered through a 0.45 micron filter and injected onto asemi-preparative HPLC column. The product, F-18-FPTP, was collected, thesolvent evaporated and the remaining dry F-18-FPTP redissolved insterile normal saline. The solution was filtered through a sterile 0.22micron filter into a sterile evacuated vial. An aliquot was removed todetermine chemical and radiochemical purity by analytical HPLC. Thespecific activity was also determined at this time.

Synthesis of F-18-fluoropropyltriphenyl phosphonium ion

Results: The biodistribution of the tracers is presented in Table 1. Ourcomparative biodistribution studies in mice indicate that F-18-FBnTP acteven better than C-11-TPMP as a PET perfusion agent. F-18-FBNTP uptakein heart is significantly greater than the other tracers, even that ofC-11-TPMP, whereas clearance from blood is as good as that of theC-11-TPMP. Many investigators have reported that apoptosis plays asignificant role in acute myocardial infarction and the pathogenesis ofother forms of heart failure. These data suggest that F-18-FBnTP has apotential utility to assess myocardial diseases including apoptosis inconnection with the MMP function as well as the myocardial perfusiontracer.

TABLE 1 Biodistribution of fluorophosphonium cations in mice 60 minafter iv. injection in comparing with various tracers. [18F]FBnTP[18F]FPTP [11C]TPMP [³H]TPP [^(99m)Tc]MIBI blood 0.02 ± 0.00 0.01 ± 0.000.03 ± 0.00 0.09 ± 0.02 0.26 ± 0.05 brain 0.07 ± 0.01 0.03 ± 0.00 0.06 ±0.02 0.06 0.02 0.08 ± 0.04 heart 35.39 ± 5.02  9.86 ± 0.82 13.4 ± 0.9520.7 ± 1.92 5.08 ± 0.25 lung 7.38 ± 1.55 2.02 ± 0.34 2.20 ± 0.21 2.81 ±0.41 0.98 ± 0.19 liver 3.00 ± 1.12 4.22 ± 1.79 5.68 ± 0.60 6.12 ± 1.085.55 ± 1.08 spleen 1.79 ± 0.57 1.23 ± 0.26 1.43 ± 0.18 1.93 ± 0.47 1.27± 0.33 kidney 4.89 ± 0.88 5.74 ± 0.54 3.79 ± 0.83 4.37 ± 0.66 12.6 ±2.57 muscle 4.31 ± 1.30 2.06 ± 0.53 2.49 ± 0.35 2.22 ± 0.41 1.56 ±0.31 * = % ID/g

Example 13 Differentiation of Tumor From Inflammation UsingF-18-Phosphonim Cations

The carcinogen nitrosomethyl urea (NMU) induces carcinoma tumors solelyin the mammary gland of female rats. Therefore, the orthotopic NMUmammary tumor is an excellent model for evaluating tracer tumorselectivity by contrasting radioactivity accumulating in mammary glandinfested with carcinoma cells and healthy mammary gland. Freund CompleteAdjuvant (FCA) is a well_studied inflammation agent in rats Induction ofFCA inflammation in NMU-bearing rats allows for direct quantitation oftracer capability to differentiate carcinoma from inflammation.

Methods:

0.1 ml of nitrosomethyl urea (NUM) was introduced i.p in female rats(150 g). When tumor reached an approximate size of 1 to 1.5 cm, FCA wasinjected (0.15 ml) to the hind limb footpad. Uptake assays were carriedout 3 days thereafter. F-18-FBnTP (0.25 mCi) was injected via tail vein.Sixty minutes later tumor, inflamed tissue and healthy muscle tissue ofthe opposite limb (control) were collected on ice, weighed and countedin a gamma counter together with standards.

Results: FIG. 3 depicts F-18-FBnTP activity in malignant mammary gland(Tumor), healthy mammary gland (Control gland), inflammation site(Inflammation), normalized to muscle. F-18-FBnTP uptake in tumor is4-times greater than that in healthy gland, and 3-times greater than ininflammation. F-18-FBnTP differential uptake in mammary gland carcinomaversus healthy gland and inflammation muscle is a good evidence for theefficacy of the invention.

The accumulation of the novel F-18-phosphonium cations, F-18-FBnTP andF-18-fluoropropyl triphenyl phosphonium (F-18-FPTP) in comparison withF18-FDG in FCA-induced inflammation and healthy tissue is presented inTable 2. Induction of inflammation and activity counting were carriedout as described above.Fluorophosphonium compounds accumulate in inflammation much less thanFDG (Table 2). These date provide strong evidence to our claim thatF-18-phosphonium compound are suitable for differentiation of tumor frominflammation and may resolve a major drawback of F-18-FDG.Table 2: Accumulation of Fluorophosphonium Compounds (FBnTP and FPTP)FDG in inflammation tissue and healthy muscle 3 days afteradministration of FCA.

TABLE 2 Accumulation of fluorophosphonium compounds (FBnTP and FPTP) FDGin inflammation tissue and healthy muscle 3 days after administration ofFCA. and Inf/ Blood Inflammation Control Control [¹⁸F]FPTP *0.037 ±0.01  0.07 ± 0.01 0.29 ± 0.06 0.25 ± 0.04 [¹⁸F]FDG 0.17 ± 0.06 1.20 ±0.16 1.05 ± 0.35 1.20 ± 0.29 [¹⁸F]FBnTP 0.10 ± 0.01 0.27 ± 0.22 0.76 ±0.21 0.42 ± 0.40 [¹⁸F]FDG 0.40 ± 0.14 1.42 ± 0.53 0.68 ± 0.14 2.12 ±0.73 *= % ID/g tissue

Example 14 Detection of Response of Lung Carcinoma Tumor to theChemotherapy Agent Taxeter_In Vivo

Methods: 2×106 human lung carcinoma A549 cells were inoculated s.q. in12 nude mice. When tumor size reached an approximate size of 5_(—)10 mm,six mice were injected i.v. with taxeter and six mice served as control.Uptake assays were carried out 48 hours thereafter. 25 μCi of F-18-FBnTPwere injected i.v., and tumor and muscle tissue was dissected after 60min.

Results: FIG. 5 depicts tumor activity normalized to muscle. Taxeterproduced a nearly 50% decrease in F-18-FBnTP accumulation in tumor,compared to non-treated mice.

Example 15 Detection of Apoptosis Induced by Androgen Depletion

Methods: Male rats were castrated and uptake assays were conducted 4days thereafter. The ventral (VP), anterior (AP) and the dorsolateral(DLP) lobes of the prostate were dissected together with hind limbmuscle. After counting tracer activity in the tissue samples, TUNELstaining was carried out and the fractions of apoptotic cells in theventral and anterior lobes were measured.

Results: Data in FIG. 6 are the mean of 9 treated and 8 control rats,normalized to muscle. Castration induced a lobe-specific decrease in theventral aspects, but not in the anterior and the dorsolateral lobes.These finding are in line with fraction of apoptotic cells, as countedin the stained histological section. In the VP lobe, 12.4%±3.8% of cellsdemonstrated DNA laddering (FIG. 8), compared to only 3.3%±1.7% of cellsin the AP lobe (FIG. 9).

Example 16 F-18-FBnTP Selectivity for Prostate Carcinoma

The data presented above show that F-18-PhC is capable of detecting theapoptotic process it the whole animal. In the prostate, moreover,alterations in F-18-PhC accumulation correlate with extent of apoptosisin the target tissue. These data suggest suitability of F-18-PhC as aPET tracer for measuring the efficacy of chemotherapy in prostatecarcinoma and most probably in other type of carcinomas as well.However, the capability of a tracer to accurately report of extent ofapoptosis may depends on the selectivity of the tracer to the tumor. Toaddress this question, tumor selectivity of F-18-FBnTP was studied inorthotopic model of prostate carcinoma.

Methods: 2×106 cells were injected, under anesthesia, into the prostateepithelial tissue of nude mice. When tumor reached an approximate sizeof 5 mm, uptake assays were performed as described above. Tumorselectivity of F-18-FBnTP and F-18-FDG was compared (3 mice in eachgroup)

Results: FIG. 9 depicts the accumulation of tracer in the normal andmalignant prostate, normalized to muscle. The uptake ratio ofmalignant-to-normal prostate tissue was 2.5 for F-18-PhC and 1.25 forFDG. This data provides further support for the suitability of F-18-PhCto detect prostate carcinoma and to measure response to treatment.

Example 17 C-11-TPMP Uptake Kinetics in the Myocardium

We have examined the performance of C-11-triphenyl phosphonium cation(TPMP) for assessing regional myocardial flow in dog using PET (Kraus,1994).

Methods: Four mCi of C-11-TPMP were introduced i.v., and dynamic imagesof increasing duration (15 sec to 20 min) were acquired over a totaltime of 85 min. Images were acquired on the GE 4096+ PET scanner (15slices, 6.5 mm slice thickness). Images were reconstructed usingbackprojection, and corrected for attenuation. A detailed description ofmethods appears in Kraus, 1994.Results: Axial sections of the heart at 5, 30 and 60 min after injectionare shown in FIG. 10. These images show excellent visualization of themyocardium with a high contrast to the surrounding lung tissue. Duringthe plateau time period heart/lung ratio was >14:1, and heart/blood>100:1.The extraction fraction of C-121-TPMP in the dog heart as a function ofmyocardial blood flow is shown in FIG. 11. Under baseline conditions(flow=69 ml/min/100 g), the extraction fraction is very high (91%). Afive-fold increase (by adenosine) in flow resulted in a 39% decrease ofthe extraction fraction.To investigate the relationship between myocardial blood flow andC-11-TPMP uptake in the heart, the LAD was occluded and the C-11-TPMPaccumulation in tissue samples compared to microsphere determinedregional myocardial blood flow. A significant correlation (r=0.93,p<0.01) was found at 5 min after LAD occlusion (FIG. 12). Thenon-infracted/infracted myocardium ratio was 12.1±2.4.These data point out the excellent features of the phosphonium cation asa perfusion agent for assessing myocardial blood flow, compared to othercurrently used SPECT perfusion agents. Thallium 201 extraction underbaseline flow is about 80%, decreasing to about 60% for five-foldincreases in flow rate. MIBI extraction for a normal flow is about 60%,decreasing to 40% for high flow rates. The advantages of C-11-TPMP PETtechnology over these SPECT agents are: (1) an overall higher myocardialextraction of C-11-TPMP; (2) a prolonged retention of C-11-TPMP in themyocardium; and (3) the better temporal and spatial resolution of thePET scanner for better documentation of ischemic regions in themyocardium.

Example 18 F-18-FBnTP Uptake Kinetics in the Myocardium

Methods: Mongrel dogs (BW=35 kg) were injected with 3-4 mCi ofF-18-FBnTP. Images were acquired on a GE 4096+ scanner (15 slices, 6.5mm slice thickness). PET scans of increasing duration (15 sec to 20 min)were acquired over a total time of 85 min post-injection. Arterial bloodsamples (0.5 ml in volume) were collected every few seconds for thefirst 3 minutes and at gradually increasing intervals (1 to 10 min) forthe remaining time of the imaging study. The vascular and myocardialkinetics of F-18-FBnTP was analyzed using the ROI method.

Results: F-18-FBnTP demonstrated a rapid washout from the blood pool(FIG. 13). F-18-FBnTP accumulated rapidly in the myocardium, reachedequilibrium within a few minutes which persisted for the remainingscanning time (FIG. 14). Rapid uptake was seen in both the left andright ventricle (FIG. 15). In contrast, F-18-FBnTP demonstrated rapidclearance from the atrium as well as from the adjacent lungs (FIG. 15).At 60-85 min post-injection period the ratio of myocardium to atrium andto lung was >15:1. Consequently, F-18-FBnTP afforded high-contrastcardiac images of an excellent visual clarity (FIG. 16).

Example 19 F-18-FBnTP Myocardial Accumulation in Heart Failure

Pacing of the mongrel dog heart at high rate (210 bpm) for a four weeksis a well-established model of heart failure. The advantage of thismodel is that the cardiomyopathy solely involve apoptosis withoutstenosis of coronary artery or related ischemia. Therefore, in thismodel the affect of apoptosis on FBnTP uptake can be dissected.

Methods: Preparation of dog and data acquisition was performed asdescribed above. Mongrel dog was installed with a pacemaker in the ribcase and underwent FBnTP PET scan (presented above). Following thebaseline scan, the dog_s heart was paced at a rate of 210 bpm for 4weeks and a second scan was acquired.

Results: FIG. 17 depicts the short-axis images of FBnTP before and after4-wks pacing. Pacing produced a significant decease in FBnTP uptake of40-60% throughout most of the inferior wall (FIGS. 18-19, Table 3).Despite that the PET scan was not gated, but due to the superb perfusioncapacities of FBnTP and consequently excellent clarity of the myocardiumleft ventricular wall, pacing-induced remodeling of the myocardiumtypical to heart failure, including thinning of the left ventricularwall and dilation of the left ventricular chamber can be seen clearly.

Moreover, pacing induced a significant decrease in the accumulation ofF-18-FBnTP throughout the entire inferior wall, indicating an enhancedprocess of heart failure mediating apoptosis of myocytes in thissegment. FIG. 18 depicts co-registered images before and after pacing. Asignificant decrease in F-18FBnTP uptake is seen in the inferior wall.Quantitation of FBnTP in the myocardium was performed using region ofinterest placed on co-registered myocardial images, whose activity wasnormalized to the injected dose, before and after pacing. An example forROI placement is illustrated in FIG. 18.Myocardial pacing induced a significant (p<0.001) decrease of 40 to 60%in the inferior wall (see table 3)

TABLE 3 F-18-FBnTP myocardial uptake before and after pacing. Forpositioning of ROI see FIG. 18. Data were derived as illustrated in FIG.19 Baseline 4-Wks Pacing Ratio P/B Slices 46-49 ROI1 *108.62 60.70 0.56ROI2 118.89 90.93 0.76 ROI3 119.11 97.86 0.82 Slices 36-37 ROI1 72.2244.46 0.62 ROI2 89.40 81.06 0.91 ROI3 102.46 86.93 0.85 *averageactivity (% of injected dose) accumulated over the over 36 to 85 min perslice, mean of activity of denoted coronal slices. Position of ROIs asdepicted in the above upper left image. Same ROIs template was used toretrieve all data.

The disclosures of all articles and references mentioned in thisapplication, including patents, are incorporated herein by reference.

The invention and the manner and process of making and using it, are nowdescribed in such full, clear, concise and exact terms as to enable anyperson skilled in the art to which it pertains, to make and use thesame. It is to be understood that the foregoing describes preferredembodiments of the present invention and that modifications may be madetherein without departing from the spirit or scope of the presentinvention as set forth in the claims. To particularly point out anddistinctly claim the subject matter regarded as invention, the followingclaims conclude this specification.

What is claimed is:
 1. A method of imaging cardiac infarction, cardiacperfusion, heart failure, cardiomyopathy or ischemia comprising:providing a radiolabeled salt comprising at least one pharmaceuticallyacceptable anion and ¹⁸F-4-fluorobenzyl-triphenylphosphonium ion;contacting cells or tissues in a subject with the radiolabeled salt; andmaking a radiographic image.
 2. The method of claim 1, wherein the imagecorrelates with mitochondrial membrane potential (ΔΨm).
 3. The method ofclaim 2, wherein the image correlates with suppressed or enhancedapoptosis.
 4. The method of claim 1, wherein the image correlates withimaging mitochondrial dysfunction.
 5. The method of claim 1, wherein theradiolabeled salt exhibits a target to non-target ratio of at leastabout 5:1.
 6. The method of claim 1, wherein the radiolabeled salt isstable in vivo.
 7. The method of claim 1, wherein the radiolabeled saltsubstantially localizes to a site or sites having dysfunctionalmitochondria within about 120 minutes after administration.
 8. Themethod of claim 1, wherein the radiolabeled salt substantially localizesto a site or sites dysfunctional mitochondria within about 60 minutesafter administration.
 9. The method of claim 1, wherein the radiolabeledsalt substantially localizes to a site or sites dysfunctionalmitochondria within about 30 minutes after administration.
 10. Themethod of claim 1, wherein the radiolabeled salt is detected by a gammacamera, positron emission tomography (PET) or single photon emissiontomography (SPECT).
 11. The method of claim 1, wherein the subject is ahuman, rat, mouse, cat, dog, horse, sheep, cow, monkey, avian, oramphibian.
 12. The method of claim 1, wherein the cell is a myocyte. 13.The method of any one of claims 7-9, wherein the site or sites are inthe myocardium.